DE2447805A1 - METHOD FOR MANUFACTURING SYNTHETIC L-PYROGLUTAMYL-L-HISTIDYL-L-PROLINAMIDE - Google Patents

Description

KRAUS & VVEISERT

PATENT LAWYERS

DR. WALTER KRAUS DIPLOMA CHEMIST. DR.-ING. ANNEKÄTE WEISERT DIPL.-tNG. SPECIALIZATION CHEMISTRY D-8 MÜNCHEN 19 · FLÜQGENSTRASSE 17 · TELEFON Ο89 / 17 7ΟΘ1

1049 / WK / jo

Istituto Farmacologico Serono S.p.A., Rome, Italy

Process for the production of synthetic L-pyroglutamyl L-halostidyl-L-prolinamide

The thyrotropin releasing hormone (TRH) or releasing factor is the hypothalamus factor, which controls the release of the thyroid-stimulating hormone (thyrotropin) stimulated from the anterior pituitary lobe.

It has recently been shown that the TRH, the first has been isolated from sheep hypotalami and then from porcine hypotalami, the structure of L-pyroglutamyl-L-histidyl-L-prolinamide Has:

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-CO-NH-CH -CO- N

(I) CH ₂

CONH

(hereinafter referred to as pGlu-His-Pro-NH ₂ ).

It has also been shown that synthetic TRH has the same hormonal activities as porcine TRH and that both porcine TRH and synthetic TRH releases thyrotropin from the pituitary gland of normal humans.

Synthetic TRH is therefore used for research on the role of TRH as a control factor in normal and abnormal secretion of thyrotropin and the influence of TRH on the secretion of the other pituitary hormones very valuable. Several methods have been developed to produce synthetic TRH. Most, however, require the use of parent protected amino acids and / or intermediate dipeptides and they have lengthy and troublesome steps of deprotection.

On the other hand, if unprotected amino acids are coupled with each other and with dipeptides, then are the yields are very low.

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In view of the fact that at least two theoretical coupling steps are required to produce the desired tripeptide, it will be understood that, based on starting materials _9, only an intolerably low final yield is obtained. In assessing the overall economics of the process, it must also be taken into account that rigorous precipitation and purification steps are required to isolate the desired intermediate and end products from the reaction mixtures, which inevitably contain significant amounts of by-products.

There is therefore a need for a new, quick one and economical processes for the synthesis of high purity TRH in high yields.

The invention now produces highly pure synthetic TRH with high yield by first adding the protected tripeptide pyroglutamyl-N ^m -benzylhistidylprolinamide of the formula:

^ .J-CO-NH-CH-CO-N

(hereinafter referred to as PGIu- (BzI) HiS-PrO-NH ₂ ) produces this by means of sodium in liquid ammonia

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reductively converted into the unprotected form and that one passes the resulting crude unprotected tripeptide through a gel filtration device.

The protected tripeptide is preferably prepared according to the invention by coupling Nt-butyloxycarbonyl-N benzylhistidine with prolinamide in the presence of N, N ¹ -dicyclohexylcarbodiimide, whereby the protected dipeptide Nt-butyloxycarbonyl-N ^m -benzylhistidyl-prolinamide is obtained in high yield.

Removal of the t-butyloxycarbonyl group by glacial acetic acid saturated with HBr yields the N -benzylhistidylprolinamide dihydrobromide, which is purified by recrystallization and reacted with an active ester of pyroglutamic acid to form the protected tripeptide PGIu- (BzI) HiS-PrO-NHp. Alternatively, which is less preferred, N-carbobenzoxypyroglutamic acid and the benzyl ester of N ^m -benzylhistidine can also be coupled by the mixed anhydride process and the resulting dipeptide ester converted into pyroglutamyl-N ^m -benzylhistidine by selective hydrogenolysis. The protected dipeptide is then reacted with prolinamide in the presence of Ν, Ν'-dicyclohexylcarbodiimide to pGlu- (Bzl) His-Pro-NH ₂ .

The reductive removal of the protecting group of the protected tripeptide according to the present invention is carried out carried out by dissolving the tripeptide in liquid ammonia, followed by gradually adding sodium to the solution is given. The reaction time is counted starting from the moment when the solution turns a steady blue Color takes on. After an extremely short period of

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the reaction is terminated by about 30 to 60 seconds glacial acetic acid is carefully added.

The response time is a bit critical. Periods of less than 30 seconds may not be sufficient to to achieve an essentially complete conversion of the tripeptide into the unprotected form. on the other hand Periods of more than 60 seconds can cause the product to decompose.

It is known that reduction with sodium in liquid ammonia is a somewhat drastic method. As such, this reaction has never been described or suggested as a suitable way to convert protected synthetic TRH into the unprotected form. Surprisingly, it has now been found that the protected tripeptide pGlu- (Bzl) His-Pro-NH ₂ does not undergo the reductive deprotection step undergoes substantial decomposition of the tripeptide molecule.

Another surprising feature of the invention lies in the great ease with which the tripeptide converted into the unprotected form is chromatographed can be cleaned. Just a single pass of the reaction product converted into the unprotected form through a chromatographic column packed with a suitable gel filtration device, delivers extremely pure synthetic TRH directly in high yield.

The corresponding eluate fractions that the desired Product are easily recovered due to the sharp and symmetrical shape of the peak.

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In contrast, the final cleaning steps in the processes according to the state of the art are lengthy and complex, if you found with the inventively erfoi compares the single filling level.

The invention therefore provides a simple method which overcomes the main disadvantages of the prior art, i.e., the problem of making undecomposed, essentially completely, unprotected form transferred TRH as well as the previously required complex Final cleaning of the product.

Examples of suitable gel filtration media are polyacrylamide, dextran and agarose gel. It has shown, that such dextran gels, such as the products with the trademark Sephadex (a range of pearl-shaped dextran gels, which is manufactured by Pharmacia, Uppsala, SwedenX are very well suited to the final cleaning stage of the peptides in the method of the invention.

Synthetic TRH is usually derived from the L-forms of the Starting amino acids made to match the structure of natural TRH. In the Examples and in the claims therefore refer to the terms "pyroglutamic acid", "histidine" and "Proline" and those of its derivatives or abbreviations to the L-forms of the corresponding amino acids or their derivatives.

However, it is within the scope of the invention to use either the L or the D or both forms.

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The invention is illustrated in the examples. example 1

Prolinamide hydrochloride (6g) and Nt-butyloxycarbonyl-N ^m - "benzylhistidine (13.8 g) were reacted in 200 ml of acetonitrile in the presence of 4.04 g of triethylamine and 10.3 g of N, N ¹ -dicyclohexylcarbodiimide, whereby Nt-butyloxycarbonyl -N ^im -benzylhistidyl-prolinamide was obtained as an oily residue. The oil was dissolved in 150 ml of 2N hydrobromic acid solution in glacial acetic acid. The solution was left in a plugged flask at room temperature for 1 hour and then poured into 1500 ml of anhydrous ether. The solid precipitate was redissolved in methanol and reprecipitated with ether, yielding 13.2 g (65.696) of crystalline N ^im -benzylhistidyl-prolinamide dihydrobromide, the melting point after recrystallization from methanol ether being 196-198 ° C.

A suspension of 13.2 g of the above protected dipeptide dihydrobromide in 150 ml of N, N-dimethylformamide containing 5.29 g of triethylamine was mixed with a solution of 9.9 g of pentachlorophenyl pyroglutamate in 130 ml of N, N-dimethylformamide. After one hour of vigorous ^{stirring at 0 to 5 °} C. and subsequent stirring for 24 hours at room temperature, the reaction mixture was evaporated to dryness in vacuo.

The residue was dissolved in 100 ml of distilled water. The solution was filtered off and the aqueous The filtrate was again evaporated to dryness in a high vacuum.

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evaporated to give an oily residue which was redissolved in a mixture of 45 ml of ethyl acetate and 10 ml of methanol.

After filtering off the triethylamine hydrobromide precipitate, evaporating the filtrate to dryness in vacuo, Washing the oily residue with 100 ml of anhydrous ether, filtering and drying gave 8.24 g (69.596) crystalline pGlu- (BzI) His-Prο-NHp recovered.

The product gave in the thin-layer analysis on silica gel G using a 60s45:20 mixture Chloroform, methanol and 30% acetic acid as eluents a single spot (R- = 0.76).

Example 2

3.1 g of N-carbobenzoxy-pyroglutamic acid were dissolved in 80 ml of tetrahydrofuran and 2 ml of triethylamine and 1.27 ml of ethyl chloroformate were added to the solution while stirring. The reaction mixture was kept ^{at 0 ° C. during the addition.} It was then left to stand at room temperature for 1 hour. To the above mixture, a solution of 8 g of N -benzylhistidine benzyl ester dibenzenesulfonate and 4 ml of triethylamine in 40 ml of tetrahydrofuran was slowly added and the whole was reacted for about 4 hours.

After evaporating to dryness in vacuo, washing the residue with 100 ml of water, extracting twice with 100 ml of methylene chloride, drying on anhydrous Na ₂ SO ^, filtering and evaporating to dryness in vacuo, the residue was dissolved in methanol and

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precipitated with anhydrous ether. This gave the crystalline N-carbobenzoxy-pyroglutamyl-N ^m -benzylhistidine benzyl ester with a melting point of 163 to 165 ° C. The yield is 4.2 g (61.4%).

4.2 g of the above protected dipeptide was dissolved in 300 ml of ethanol. The mixture was made with 0.5 g of 10% palladium on carbon and it was hydrogenated for 3 hours at room temperature under a hydrogen pressure of 2 atm. Crystalline pyroglutamyl-N -benzylhistidine was obtained in a yield of 2.19 g (85%) as the reaction mixture of the hydrogenation conventional procedures such as filtration, evaporation and precipitation with anhydrous ether from an ethanolic solution of the residue, was subjected (for redissolution of the residue, 30 ml of ethanol were used).

2.19 g of N-pyroglutamyl ⁱⁿ -benzylhistidin, 0.93 g Prolinamidhydrochlorid and 2 ml of triethylamine were dissolved in 50 ml Ν, Ν-dimethylformamide, followed by cooling to 0 ⁰ C and a solution of 1.52 g Ν, Ν ' -Dicyclohexylcarbodümid was implemented in 20 ml of N, N-dimethylformamide.

After stirring for 48 hours at room temperature, filtering off and evaporating to dryness, 1.25 g (45%) pGlu- (BzI) HiS-PrO-NH _{2 were} obtained and purified by recrystallization from methanol.

Thin layer chromatography indicated that the product appeared to be identical to the product of Example 1.

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Example 3

A nitrogen-protected reactor equipped with a magnetic stirrer wa2% was charged with about 500 ml of liquid ammonia and 1 g of pure PGIn- (BzI) HiS-PrO-NH ₂ . Sodium was gradually added until the solution took on a permanent blue color. After 30 seconds, the reaction was terminated by carefully adding about 1 ml of glacial acetic acid. The mixture was evaporated to dryness and the residue was redissolved in 0.2N acetic acid.

The resulting solution was filtered off and passed through a chromatography column measuring 2.2 × 110 cm, which was packed with Sephadex G-15 which had been equilibrated with 0.2N acetic acid. Carrying out the gel filtration at 25 ml / h using the same 0.2N acetic acid as the eluent gave a symmetrical peak of the eluate. The eluate fractions corresponding to the peak area were collected and lyophilized to give 0.58 g (72.5%) of high purity pGlu-HiS-PrO-NH ₂ (TRH). A thin layer analysis carried out on plates with silica gel G, which had been activated for one hour at 120 ° C (250 μ), using either the system chloroform: methanol: concentrated ammonia (60: 45: 20) or the system chloroform: methanol: 3096 acetic acid (60: 45: 20) was carried out, showed a single spot in both cases with R _f values of 0.6 and 0.36, respectively.

The product appeared to be completely free of ammonium (Nessler's reagent test) and halide ions.

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The amino acid analysis confirmed the expected structure,

d ° «- 41 ° (c« ΐ in methanol). Example 4

Example 3 was repeated with the exception that 8.24 g of pure pGlu- (BzI) HiS-Prο-NH ₂ were converted into the unprotected form with sodium in 2500 ml of liquid ammonia and that about 6 ml of glacial acetic acid were used to to stop the reaction.

The crude reaction product was passed through a 5.5 x 135 cm column of Sephadex G-15 at ml / hr using the same eluant as in Example 3. 4.65 g (70.5%) of high-purity pGlu-His-Pro-NH _{2 were} obtained.

The analysis results showed that this product was extremely pure and that it was the same as with both products of the example 3 was identical.

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Claims (5)

Claims 1. Process for the preparation of high purity pyroglutamylhistidylprolinamide (TRH) by synthesis from protected amino acids, removal of protecting groups and final cleaning of the raw? in the unprotected form converted tripeptides, characterized in that pyroglutamyl-N -benzylhistidyl-prolinamide is reductively using sodium in liquid ammonia transferred into the unprotected form and that one the raw passes unprotected tripeptide through a gel filtration device. 2. The method according to claim 1, characterized in that the reductive conversion in carries out the unprotected form in such a way that the protected tripeptide is dissolved in liquid ammonia, gradually adding sodium until the solution takes on a permanent blue color, and the reaction with glacial acetic acid 30 to 60 seconds after the permanent blue color appears. : 3. The method according to claim 1 or 2, characterized in that the raw, in the Unprotected form converted tripeptide is recovered from the reaction mixture and without an intermediate purification step passes directly through the gel filtration device. 4. The method according to any one of claims 1 to 3, characterized in that as Gel filtration device used dextran gel. -13- 50981 7/1211 5. The method according to any one of claims 1 to 4, characterized in that the Purifies raw tripeptide converted into the unprotected form by means of a single gel filtration stage. 5 0 9 8 17/1211